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The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/β-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment. As part of The Cancer Genome Atlas Pan-Cancer effort, data analysis for point mutations and small indels from 3,281 tumours and 12 tumour types is presented; among the findings are 127 significantly mutated genes from cellular processes with both established and emerging links in cancer, and an indication that the number of driver mutations required for oncogenesis is relatively small. Genomic landscape of twelve tumour types As part of The Cancer Genome Atlas Pan-Cancer project, these authors present data analysis for point mutations and small indels from more than 3,000 tumours representing 12 tumour types. Among the findings are 127 significantly mutated genes from cellular processes with both established and emerging links to cancer, and an indication that the number of driver mutations required for oncogenesis is relatively small. Additional analyses also identify genes with significant impact on survival and a likely temporal order of mutational events during tumorigenesis.

Siyuan Zheng, Roel Verhaak and colleagues report an analysis of telomere lengths and somatic alterations in telomere-related pathways across 31 cancer types. Their study provides an overview of the molecular mechanisms driving TERT expression and activation of the ALT pathway, and identifies a subset of tumors with neither detectable TERT expression nor somatic alterations in ATRX or DAXX . Cancer cells survive cellular crisis through telomere maintenance mechanisms. We report telomere lengths in 18,430 samples, including tumors and non-neoplastic samples, across 31 cancer types. Telomeres were shorter in tumors than in normal tissues and longer in sarcomas and gliomas than in other cancers. Among 6,835 cancers, 73% expressed telomerase reverse transcriptase (TERT), which was associated with TERT point mutations, rearrangements, DNA amplifications and transcript fusions and predictive of telomerase activity. TERT promoter methylation provided an additional deregulatory TERT expression mechanism. Five percent of cases, characterized by undetectable TERT expression and alterations in ATRX or DAXX , demonstrated elongated telomeres and increased telomeric repeat–containing RNA (TERRA). The remaining 22% of tumors neither expressed TERT nor harbored alterations in ATRX or DAXX . In this group, telomere length positively correlated with TP53 and RB1 mutations. Our analysis integrates TERT abnormalities, telomerase activity and genomic alterations with telomere length in cancer.

Emerging data indicate that alterations in cytokine synthesis may play a role in inflammatory bowel disease (IBD) pathogenesis. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. The aim of this study was to assess the relationship between polymorphisms involving five cytokine genes (TNF-alpha, TGF-beta, IL-10, IL-6, and IFN-gamma), and IBD susceptibility and disease phenotype. Cytokine genotyping was performed utilizing polymerase chain reaction. The specific gene polymorphisms that were probed for included: -1082(G/A), -819(T/C), and -592(A/C) in the IL-10 promoter, -308(G/A) in the TNF-alpha promoter, codon 10 (T/C), and codon 25 (G/C) of the TGF-beta signal sequence, +874(T/A) of intron 1 of IFN-gamma, and -174(C/G) in the IL-6 promoter. A total of 193 IBD patients (138 Crohn's disease (CD) and 55 ulcerative colitis (UC)) and 92 controls were evaluated. No association between IBD, UC, or CD susceptibility and the cytokine gene polymorphisms were found. Patients with ileocolonic CD were more likely to possess the IL-6 -174 GG genotype compared to those with nonileocolonic disease (p= 0.006). Patients with ileal CD were more likely to possess the IL-6 -174 GC genotype compared to those with nonileal disease (p= 0.0004). An increased number of CD patients with isolated colonic disease possessed the IL-6 -174 CC genotype compared to those with nonisolated colonic disease (p= 0.032). The cytokine gene polymorphisms studied here do not appear to influence IBD susceptibility. There does, however, appear to be an influence on disease phenotype, particularly on CD site.

CRISPR/Cas9 DNA editing creates a double-stranded break in the target DNA, which can frequently generate random insertion or deletion of bases (indels); a new genome editing approach combining Cas9 with a cytidine deaminase is described here, which corrects point mutations more efficiently than canonical Cas9, while avoiding double-stranded breaks and indel formation. DNA edits without double-helix breakage The CRISPR/Cas technology widely used for genome editing involves formation of a double-strand break in the target DNA sequence. When used to modify a single nucleotide, this procedure frequently generates DNA insertions or deletions (indels). David Liu and colleagues describe an approach that obviates DNA cleavage, as a means to avoid such off-target mutations. This 'base editing' method, which utilizes a composite enzyme consisting of CRISPR/Cas9 and the APOBEC1 deaminase, can directly convert C to T (or G to A). They also describe modifications that increase the yield of the desired correction and significantly suppressing indel formation. Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction 1 , 2 . Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks 1 , 2 . Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

Targeted editing of single base pairs is achieved in monocot rice and dicot tomato using Target-AID (Cas9 activation-induced cytidine deaminase fusion). We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

Mutation is the ultimate source of genetic variation. The most direct and unbiased method of studying spontaneous mutations is via mutation accumulation (MA) lines. Until recently, MA experiments were limited by the cost of sequencing and thus provided us with small numbers of mutational events and therefore imprecise estimates of rates and patterns of mutation. We used whole-genome sequencing to identify nearly 1,000 spontaneous mutation events accumulated over ∼311,000 generations in 145 diploid MA lines of the budding yeast Saccharomyces cerevisiae . MA experiments are usually assumed to have negligible levels of selection, but even mild selection will remove strongly deleterious events. We take advantage of such patterns of selection and show that mutation classes such as indels and aneuploidies (especially monosomies) are proportionately much more likely to contribute mutations of large effect. We also provide conservative estimates of indel, aneuploidy, environment-dependent dominant lethal, and recessive lethal mutation rates. To our knowledge, for the first time in yeast MA data, we identified a sufficiently large number of single-nucleotide mutations to measure context-dependent mutation rates and were able to (i) confirm strong AT bias of mutation in yeast driven by high rate of mutations from C/G to T/A and (ii) detect a higher rate of mutation at C/G nucleotides in two specific contexts consistent with cytosine methylation in S. cerevisiae .

Single DNA base pairs are edited in wheat, rice and maize using a Cas9 nickase fusion protein. Targeted base editing in plants without the need for a foreign DNA donor or double-stranded DNA cleavage would accelerate genome modification and breeding in a wide array of crops. We used a CRISPR–Cas9 nickase-cytidine deaminase fusion to achieve targeted conversion of cytosine to thymine from position 3 to 9 within the protospacer in both protoplasts and regenerated rice, wheat and maize plants at frequencies of up to 43.48%.

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS , we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure. Synonymous mutations do not alter amino acid sequence but may exert oncogenic effects in other ways. Here, the authors present a catalogue of synonymous mutations in cancer and characterise their properties.

Mice with targeted point mutations are generated efficiently using Cas9–cytidine deaminase fusions. Base editors (BEs) composed of a cytidine deaminase fused to CRISPR–Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44–57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.